Role of glutamine-117 in the ribonucleolytic activity of human angiogenin.

The crystal structure of human angiogenin (reported in the preceding paper in this issue) reveals that the site that corresponds to the pyrimidine binding site of RNase A is obstructed by Gln-117. Mutation of this residue to Ala and Gly is here found to increase activity 11- to 18-fold and 21- to 30-fold, respectively, toward dinucleotide, polynucleotide, and cyclic nucleotide substrates, but without changing specificity. The enhanced activity of Q117G toward CpA is due to a 5-fold decrease in Km and a 6-fold increase in kcat. Its Ki value for 2'-CMP is 5-fold lower than that of native angiogenin, whereas its Ki value for 5'-AMP is unchanged. It has been reported previously that mutating Asp-116 to Ala increases activity 15-fold. The double mutant D116A/Q117A is shown to be only slightly more active than each individual mutant. The present results demonstrate that Gln-117 impedes the ribonucleolytic activity of angiogenin, as predicted by x-ray crystallography. Moreover, they suggest that prior to or during catalysis angiogenin must undergo a conformational change to reorient the C-terminal segment that contains this residue, and that a similar reorganization is required for the mutants as well. This view is supported by molecular modeling of an angiogenin-uridine vanadate complex. These in vitro findings have implications for the angiogenic activity of angiogenin in vivo.

本文同刊上期报道的人血管生成素（angiogenin）晶体结构显示，其对应核糖核酸酶A（RNase A）嘧啶结合位点的区域被谷氨酰胺-117（Gln-117）所遮蔽。本研究将该残基突变为丙氨酸（Ala）与甘氨酸（Gly）后发现，针对二核苷酸、多核苷酸及环核苷酸底物的酶活分别提升11~18倍与21~30倍，但并未改变底物特异性。Q117G突变体对CpA的活性增强源于米氏常数（Km）降低5倍，同时催化常数（kcat）提升6倍。其对2'-胞嘧啶单核苷酸（2'-CMP）的抑制常数（Ki）较天然血管生成素降低5倍，而对5'-腺嘌呤单核苷酸（5'-AMP）的Ki值无变化。此前有研究报道，将天冬氨酸-116（Asp-116）突变为丙氨酸可使酶活提升15倍。双突变体D116A/Q117A的活性仅略高于各单突变体。本研究结果证实，如X射线晶体学（X-ray crystallography）所预测，谷氨酰胺-117会阻碍血管生成素的核糖核酸酶活性。此外，结果提示，在催化过程中或催化前，血管生成素需发生构象变化以重定向包含该残基的C端片段，突变体亦需经历类似的构象重排。血管生成素-钒酸尿苷复合物的分子建模（molecular modeling）结果进一步支持了这一观点。上述体外实验结果对阐释血管生成素在体内的血管生成活性具有参考价值。